Suppression of Nuclear Factor-KappaB Dependent Processes Using Oligonucleotides

ABSTRACT

Antisense oligonucleotides which hybridize with nuclear factor-κB (NF-κB) mRNA and methods of using these oligonucleotides.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation application of U.S. application Ser.No. 11/888,342 filed Jul. 30, 2007, now pending; which is a continuationapplication of U.S. application Ser. No. 10/328,861 filed Dec. 24, 2002,now issued as U.S. Pat. No. 7,268,121; which is a continuationapplication of U.S. application Ser. No. 08/110,161 filed Aug. 20, 1993,now issued as U.S. Pat. No. 6,498,147; which is a continuation-in-partapplication of U.S. application Ser. No. 07/887,331, now abandoned. Thedisclosure of each of the prior applications is considered part of andis incorporated by reference in the disclosure of this application.

GRANT INFORMATION

This invention was made with Government support under Grant Nos. CA50234 and MH 47680 awarded by the National Institutes of Health. TheUnited States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods and compositions useful ininhibiting disorders dependent upon expression of the inducibletranscription factor NF-κB.

2. Background Information

The NF-κB transcription activator is a multiprotein complex which canrapidly induce the synthesis of defense and signalling proteins uponexposure of cells to a wide variety of mostly pathogenic agents. Threeprotein subunits, κB, p50, and p65, control the biological functions ofNF-κB. IκB is a 3543 kDa subunit which inhibits the DNA-binding of NF-κBand serves to retain NF-κB in an inducible form in the cytoplasm ofunstimulated cells. Upon stimulation of cells, IκB dissociates from theinactive complex with p65 and p50. The released p50-p65 complex can thenmigrate into the nucleus and potently transactivate genes.

p50, one of the two DNA-binding submits of NF-κB, serves to recognizethe more highly conserved half site in decameric sequence motifs withthe consensus sequence GGGRNNYYCC. p50 has homologies to the rel anddorsal proteins, both of which are also involved in cytoplasmic/nuclearsignalling, and is identical to factors known as KBF1, H2TF1 and EBP-1.p50 is synthesized as an inactive precursor of 110 kDA.

The other DNA binding subunit, p65, binds to the less conserved halfsite of κB motifs and is apparently also homologous to rel/dorsalproteins. The combination of p50 and p65 DNA binding subunits in NF-κBextends the repertoire of binding motifs recognized with high affinity.Only p65 appears to bind IκB. The inducibility of NF-κB is thus equallydependent on the presence of p65 as of UB. The NF-κB system is so farunique among transcription factors in that the interaction of threedistinct subunits control multiple regulatory characteristics of atranscription activator including the subcellular localization and theactivity, inducibility, and specificity of DNA binding.

Many different binding sites for NF-κB have been characterized and formost the base requirements for protein binding have been defined bymethylation interference analysis or other footprinting techniques(Baeuerle, Biochem Biophys. Act, 1072:63.1991). Most binding sites aredecameric, but some are undeca- or dodecameric. The latter may arisefrom NF-κB binding in a mutually exclusive manner to two or threedecameric motifs within the longer sequences. An alternative explanationis that p50-p65 or the p50 dimer can contact 10, 11 or even 12 basepairs. Support for the latter hypothesis comes from mutational analysisof the enhancer of the MHC class I gene H-2 K^(b).

Among those disorders which are linked to activation of NF-κB isleukemia caused by the retrovirus HTLV-1. Human T-cell leukemia virus(HTLV-1) is recognized as the etiologic agent of the human malignancy,adult T cell leukemia (B. J. Poiesz, Proc. Natl. Acad. Sci. USA,77:7415, 1980; D. J. Slamon, et al., Science, 226:61, 1984; W. C. Goh,et al., ibid 227:1227, 1985).

Circumstantial data has implicated the HTLV-I encoded tax gene inleukemo-genesis. This gene encodes a 40 kD protein that causestranscriptional transactivation of viral gene expression and alsoactivates expression of certain cellular genes that are important forgrowth (A. J. Cann, et al., Nature 318:571, 1985; B. K. Felber, et al.,Science, 229:675, 1985; J. Fujisawa, et al., Embo Journal 5:713, 1986).In vitro studies have demonstrated that tax can activate the promotersof the interleukin 2 receptor (IL-2R) a-chain, GM-CSF, fos, PDGF, IL-6,NGF, TGF-β, HIV LTR as well as its own LTR (D. J. Slamon et al.,Science, 226:61, 1984; W. C. Goh et al., ibid, 227:1227, 1985; J. Inoue,et al., Embo Journal 5:2883, 1986; S. Miyatake, et al., Mol. Cell. Biol.8:5581, 1988; K. Nagata, et al., J. Virol. 63:3220, 1989; L. Ratner,Nucleic Acid Research, 17:4101, 1989; J. Sodroski, et al., Science,228:1430, 1985; J. E. Green, et al., Mol. Cell. Biol. 11:4635, 1991; S.J. Kim, et al., J. Exp. Med., 172:121, 1990; E. Bohnlein, et al., J.Virol., 63, 1578, 1988). In vitro studies of tax effects on geneexpression, have demonstrated two independent pathways for its action ontranscription (M. R. Smith and W. C. Green, J. Clin. Invest. 87:761,1991). The first affects the family of nuclear transcription factorsrelated to c-rel which bind to NF-κB sites and are important for thenormal activation of lymphocytes. NF-κB response sequences occur in anumber of genes including the HIV LTR (G. Nable, et al., Nature London,326:711, 1987) and the IL-6 promoter (T. A. Libermann, et al., Mol.Cell. Biol., 10:2327, 1990). The heterodimer composed of the p50 and p65rel related proteins have been shown to affect the transcription of manyof these genes (P. A. Baeuerle, Biochem. Biophys. Acta, 1072:63, 1991).The other effect of tax is thought to be NF-κB independent, whereby taxactivates its own promoter through three tax responsive elements (TREs).Similar sequence motifs have been identified in fos (M. Fujii et al.,Proc. Natl. Acad. Sci. USA, 85:8526, 1988), an immediate early responsegene.

Unfortunately, although it is known that tax and other disorders appearlinked to activation of NF-κB, no therapeutic modalities exist which cansuppress this activation and thereby inhibit progression orestablishment of the disorder. The present invention addresses this needand provides composition and means of accomplishing this goal.

SUMMARY OF THE INVENTION

The present invention arose from the discovery that an antisenseoligonucleotide which hybridizes to nuclear factor-κB (NF-κB) mRNA canbe used to suppress processes which depend upon activation of NF-κB.

As a consequence of this discovery, the present invention represents amajor improvement over existing techniques for suppressing NF-κBdependent processes which often entail use of chemicals which are highlytoxic, especially in terms of their utilization in vivo. The antisenseoligonucleotides of the invention display a high degree ofbiocompatibility with host systems such that the low efficiency andtoxic aspects of prior art methodologies and compositions is avoided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, B is a Northern analysis of early genes in tax or NF-κBantisense treated cells.

FIG. 2 is a CAT assay in the presence of NF-κB p65 antisense ODNs.

FIG. 3 is an electrophoretic mobile shift assay (EMSA) of nuclearextracts obtained from unmanipulated, sense and antisense treated cells.

FIG. 4 is growth curves of cells treated with tax or NF-κB antisense, invitro. Symbols are mock (No; >), sense (SEN; □) and antisense (ANT; X).

FIG. 5 shows in vivo growth inhibition of B cell line tumors by NF-κBantisense treatment.

FIG. 6 shows the effect of antisense NF-κB antisense and N-acetylcysteine (NAC) on mouse survival after LPS challenge.

FIG. 7 shows serum IL-6 levels after LPS challenge. Error bars representone standard deviation.

FIGS. 8 a and 8 b show a Northern blot analysis of NF-κB dependent geneexpression in tissues of LPS challenged mice. Lanes 1-9 representpretreatment for 3 hours with NAC or antisense NF-κB. Lanes 10-18 showthe effects of 24 hours pretreatment.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to an antisense oligonucleotide sequencewhich can hybridize to nuclear factor κB (NF-κB) subunit mRNA. Thisantisense sequence is highly useful for suppressing in vitro or in vivoNF-κB dependent or associated processes in individuals. Such processesare typically associated with such disorders as those mediated by immuneor cytokine responses (for example, septic shock) as well as thosedisorders induced by infectious agents such as retroviruses, morespecifically, HIV and HTLV.

The antisense oligonucleotides of the invention are preferably directedto the p65 or p50 subunits NF-κB mRNAs. Most preferably, the antisenseoligonucleotides are complementary to the translation initiation nucleicacid sequence of these subunits. In general, the antisenseoligonucleotides of the invention are capable of hybridizing to DNAwhich has the nucleotide sequence 3′-TAGCAGACGGTACCACTTCTA-5′ or3′-CTTGTCAAGCAGGTACCGGC-5′ or 3′-TTTGTCTAGCAGGTACCAGT-5′. Most preferredare antisense oligonucleotides having the nucleotide sequence5′-ATCGTCTGCCATGGTGAAGAT-3′ 5′-TCGTCTGCCATGGTGAAGAT-3′, or5′-GAACAGTTCGTCCATGGCCG-3′ or 5′-AAACAGATCGTCCATGGTCA-3′. As a generalmatter, the oligonucleotide employed will have a sequence that iscomplementary to the sequence of the target RNA. However, absolutecomplementarity is not required; in general, any oligonucleotide havingsufficient complementarity to form a stable duplex with the target RNAso that translation of the RNA is inhibited, is considered to besuitable. Since stable duplex formation depends on the sequence andlength of the hybridizing oligonucleotide and the degree ofcomplementarity between the antisense oligonucleotide and the targetsequence, the system can tolerate less fidelity (complementarity) whenlonger oligonucleotides are used. However, it is presently believed thatoligonucleotides of about 8 to 40 bases in length and having sufficientcomplementarity to form a duplex having a melting temperature of greaterthan about 40° C. under physiologic conditions are particularly wellsuited for practice of the invention (Thoung, et al., PNAS USA, 84:5129,1987; Wilson, et al., Nucleic Acids Res., 16:5137, 1988). Accordingly,such oligonucleotides are preferred.

Another variable that may affect practice of the invention is the regionof the target RNA to which the selected oligonucleotide is designed tohybridize. Although oligonucleotides capable of stably hybridizing withany region of the RNA may be suitable for practice of the invention,oligonucleotides complementary to a region including the translationinitiation nucleic acid sequence of the NF-κB subunit are particularlyeffective. The antisense oligonucleotide is considered effective as longas the translation of the mRNA to which the oligonucleotide iscomplementary is inhibited.

The oligonucleotide employed may be unmodified or modified. Suitablemodifications include, but are not limited to, the ethyl or methylphosphonate modifications disclosed in U.S. Pat. No. 4,469,863 and thephosphorothioate modifications deoxynucleotides described by LaPlanche,et al. (Nucleic Acids Research 14:9081, 1986), and by Stec. et al., (J.Am. Chem. Soc. 106:6077. 1984). The modification to the antisenseoligonucleotide is preferably a terminal modification in the 5′ or 3′region. Preferred are modifications of the 3′ terminal region asdescribed herein. Furthermore, recent advances in the production ofoligoribonucleotide analogues mean that other agents may also be usedfor the purposes described here, for example, 2′-methylribonucleotides(Inoue, et al., Nucleic Acids Res. 15:6131, 1987) and chimericoligonucleotides that are composite RNA-DNA analogues (Inoue, et al.,FEBS Lett., 215:327. 1987).

Of course, in order for the cell targets to be effectively inhibited bythe selected antisense oligonucleotides, the cells must be exposed tothe oligonucleotides under conditions that facilitate their uptake bythe cells. For in vitro therapy this may be accomplished by a number ofprocedures, including, for example, simple incubation of the cells withthe oligonucleotides in a suitable nutrient medium for a period of timesuitable to achieve selective inhibition of the cells. For example,where the cell targets of the antisense oligonucleotide of the inventionare present in bone marrow cells, procedures can be employed such asthose described by Gartner and Kaplan, Proc. Natl. Acad. Sci. USA,77:4756, 1980; Coulombel, et al., Blood 67:842, 1986; Meagher, et al.,Blood, 72:273, 1988; or U.S. Pat. No. 4,721,096 with an optimalconcentration of the selected antisense oligonucleotide. After themarrow cells have been exposed to the oligonucleotide and, in somecases, cultured as described above, they are then infused into thetransplant recipient to restore haemopoiesis.

The antisense oligonucleotide of the invention can also be administeredto provide in vivo therapy to a patient having a disorder which isassociated with activation of NF-κB. Such therapy can be accomplished byadministering, in vitro and in vivo as the case may be, atherapeutically effective amount of antisense oligonucleotide, or asfurther described below, the antisense oligonucleotide in combinationwith glutathione precursor. The term “therapeutically effective” meansthat the amount of antisense oligonucleotide administered alone, or incombination with glutathione precursor, is of sufficient quantity tosuppress to some beneficial degree activation of NF-κB and the disorderassociated with activation of NF-κB. Examples of such disorders includethose associated with LPS-induced septic shock and those associated withelevated IL-6 production. The NF-κB process may also be tissue specific.For example, the disorder may be detected in the spleen, liver, kidney,or lung. Preparations utilizing antisense oligonucleotide can comprisethe oligonucleo-tide in a simple buffer solution or, alternatively, in amore complex vehicle as described below.

The invention also provides a method of monitoring the effectiveness ofsuppressing NF-κB and NF-κB dependent processes in the tissue of anindividual after administering a therapeutically effective amount ofNF-κB antisense comprising detecting the level of cytokine production ina tissue, before and after antisense therapy. Preferably, the cytokineis IL-6. IL-6 can be detected by immunological methods such as ELISA, ornucleic acid methods, such as Northern blot analysis of IL-6 mRNA.

Studies have suggested that glutathione precursors may be useful inregulating activation of NF-κB (Staal, et al., Proc. Natl. Acad. Sci.USA, §7:9943, 1990; Bruno, et al., Biochemical Pharmacology, 37:4319,1988). Typically, these precursors are an acylcysteine, such asN-acetylcysteine. These compounds appear to block activation of NF-κB inboth resting as well as activated cells. Unfortunately, these compoundshave been found to be extremely toxic in vivo when administered alone.An advantage of the present system is that it allows a means of reducingthe concentration of glutathione precursors which is utilized bycombining the precursor with the antisense oligonucleotide of theinvention. Thus, according to the method of the invention, it ispossible to administer the antisense oligonucleotide in combination witha precursor of glutathione. The term “in combination” means that theantisense oligonucleotide and the glutathione precursor are administered(1) separately at the same or different frequency using the same ordifferent administration or (2) together in a pharmaceuticallyacceptable composition. If desired, the antisense oligonucleotide andglutathione precursor can be administered substantiallycontemporaneously. The term “substantially contemporaneously” means thatthe antisense oligonucleotide and glutathione precursor are administeredreasonably close together with respect to time, for example,simultaneously to within a few hours.

“Pharmaceutical combination” includes intimate mixtures of the twocomponents of the invention, as in classical compositions, and alsonon-mixed associations, such as those found in kits or pharmaceuticalpacks.

Antisense oligonucleotide, alone or in combination with glutathioneprecursor, can be administered in a single dose or can be administeredin multiple doses over a period of time, generally by injection. Variousadministration patterns will be apparent to those skilled in the art.The dosage ranges for the administration of the antisenseobigonucleotide of the invention are those large enough to produce thedesired effect of suppressing the undesired NF-κB dependent process. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art without undue experimentation. The dosage can be adjusted by theindividual physician in the event of any counter indications, immunetolerance, or similar conditions. Those of skill in the art can readilyevaluate such factors and, based on this information, determine theparticular therapeutically effective concentration of antisenseoligonucleotide, or antisense oligonucleotide in combination withglutathione precursor, to be used. Generally, dosage for antisenseoligonucleotide can vary from about 1.0 mg/g body weight to about 100mg/g body weight, preferably from about 10 mg/g body weight to about 80mg/g body weight, most preferably from about 30 mg/g to about 50 mg/gbody weight. Glutathione precursor, such as N-acetylcysteine, can beadministered from about 1 mM to about 50 mM, more preferably from about10 mM to about 40 mM, most preferably from about 20 mM to about 30 mM.

Antisense oligonucleotide and glutathione precursor can be administeredas the compound or as a pharmaceutically acceptable salt of thecompound, alone, in combination, or in combination with pharmaceuticallyacceptable carriers, diluents, and vehicles. Most preferably, antisenseoligonucleotide and glutathione precursor are mixed individually or incombination with pharmaceutically acceptable carriers to formcompositions which allow for easy dosage preparation.

The antisense oligonucleotide and glutathione precursor composition ofthe present invention can be administered in any acceptable mannerincluding by injection, using an implant, and the like. Injections andimplants are preferred because they permit precise control of the timingand dosage levels used for administration, with injections being mostpreferred. Antisense oligonucleotide and glutathione precursorcompositions according to the present invention are preferablyadministered parenterally.

Antisense oligonucleotide according to the present invention can beadministered to the patient in any acceptable manner including orally,by injection, using an implant, nasally and the like. Oraladministration includes administering the composition of the presentinvention in tablets, suspensions, implants, solutions, emulsions,capsules, powders, syrups, water compositions, and the like. Nasaladministration includes administering the composition of the presentinvention in sprays, solution, and the like. Injections and implants arepreferred because they permit precise control of the timing and dosagelevels used for administration, with injections being most preferred.Antisense oligonucleotide is preferably administered parenterally.

Glutathione precursor according to the present invention can beadministered to the animal in any acceptable manner including byinjection, using an implant, and the like. Injections and implants arepreferred because they permit precise control of the timing and dosagelevels used for administration, with injections being most preferred.Glutathione precursor is preferably administered parenterally.

Antisense oligonucleotide and glutathione precursor compositions can beadministered in an injectable formulation containing any glutathioneprecursor and antisense oligonucleotide compatible and biocompatiblecarrier such as various vehicles, adjuvants, additives, and diluents toachieve a composition usable as a dosage form.

Aqueous vehicles such as water having no nonvolatile pyrogens, sterilewater, and bacteriostatic water are also suitable to form injectableantisense oligonucleotides and glutathione precursor formulations. Inaddition to these forms of water, several other aqueous vehicles can beused. These include isotonic injection compositions that can besterilized such as phosphate buffered saline, sodium chloride, Ringer's,dextrose, dextrose and sodium chloride, gelatin and lactated Ringer's.Addition of water-miscible solvents, such as methanol, ethanol, orpropylene glycol generally increases solubility and stability of thecompounds in these vehicles.

Nonaqueous vehicles such as cottonseed oil, squalene, sesame oil, orpeanut oil and esters such as isopropyl myristate may also be used assolvent systems for glutathione precursor and antisenseoligonucleotides. Additionally various additives which enhance thestability, sterility, and isotonicity of the composition includingantimicrobial preservatives, antioxidants, chelating agents, gelatin andbuffers can be added. Any vehicle, diluent; or additive used would,however, have to be compatible with the compounds of the presentinvention.

Antisense oligonucleotides and glutathione precursor compositionsaccording to the present invention can be administered in the form of aslow-release subcutaneous implant which is inserted beneath the skin.The implant can take the form of a pellet which slowly dissolves afterbeing implanted or a biocompatible and glutathione precursor andantisense oligonucleotide compatible delivery module well known to thoseskilled in the art. Such well known dosage forms are designed such thatthe active ingredients are slowly released over a period of several daysto several weeks.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively beadopted without resort to undue experimentation.

Example 1 Antisense Inhibition of Tumor Cells In Vitro A. Preparation ofOligonucleotides

Sense and antisense oligodeoxynucleotides (ODNs) were synthesized by thephosphoramide method on an ABI automated synthesizer (Foster City,Calif.). The phosphothioate (PS) sulfurization modification (Stein, etal., Nucleic Acids Res., 16:3209, 1988) was performed during synthesis,where TETD and acetonitrile were substituted for the usual iodine,pyridine and water during the oxidation step. ODNs were purifiedaccording to published procedures (Miller, et al., J. Biol. Chem.,255:9659, 1980).

One set of sequences was selected to be complementary to the transcriptencoded by the HTLV-I LTR-tax cassette which had previously been used togenerate transgenic mice (Nerenberg, et al., Science, 237:1324, 1987).The antisense sequence was GAAGTGGGCCATGTGGA*A*G (location 718-737 onHTLV-I LTR-tax construct) which included the AUG initiation codon(underlined) and sense sequence was CTTCCACATGGCCCACT*T*C*, the exactcomplement of the tax-antisense ODN above. Asterisks show sites of PSmodification.

An upstream sense primer (AAGCGTGGAGACAGTTCAGG, location 423-442) wassynthesized as a primer pair for the modified or unmodified antisenseprimer (location 737-718). Similarly, a downstream antisenseoligonucleotide (TTGGCGGGGTAAGGACCTTG, location 986-1004) wassynthesized to pair with the modified or unmodified sense (location718-737).

A second set of ODNs was prepared based on the nucleotide sequences ofthe p50 and p65 subunits of NF-κB. The ODNs for p50 NF-κB were antisense5′-ATCGTCTGCCATGGTGAAGAT-3′(human), antisense5′-TCGTCTGCCATGG-TGAAGAT-3′ (mouse) and sense5′-ATCTTCACCATGGCAGCAGA-3′(human and mouse). The corresponding ODNs forp65 were antisense 5′-GAACAGTT-CGTCCATGGCCG-3′ (human), antisense5′-AAACAGATCGTCCATGGTCA-3′ (mouse) and sense 5-′CGGCCATGGACGAACTGTTC-3′(human) and sense 5′-TGACCATGGACGATCTGTTT-3′(mouse). Initiation codonsare underlined. Both sets of ODNs extended over the translationalinitiation sites of tax or NF-κB mRNAs.

B. Establishment of Tax Expressing Tumor Cell Lines

LTR-tax transgenic mice were originally generated from CD1 females and(C57BL/6×DBA/2) F1 males, and thus were genetically highly heterogeneous(Nerenberg, et al., Science, 237:1324, 1987). One line (6-2) containing40 copies of the transgene and expressing high levels of tax was backbred to C57BL/6 mice for greater than 30 generations. Spontaneouslyarising fibroblastic tumors were sterily harvested, finely minced andgrown in bulk culture for 30 passages. Single cell clones were obtainedby dilution and growth in Terasaki plates in the presence of filteredconditioned medium from the bulk cultures. Individual clones were grownin bulk culture and tested for ability to form tumors in syngeneicanimals. One of these lines (designated B line) was selected for furtherstudy as it rapidly and reproducibly caused tumors when innoculated intothe hind limb of animals when 1×10⁶−1×10⁷ cells were injected. Tumorsgrew rapidly at the site of local injection and never caused metastasis.These tumors attracted high numbers of granulocytes, similar to theoriginal tumors (Nerenberg, et al., Science, 237:1324, 1987), presumablycaused by high secretion of cytokines. Mice typically die from localizedeffects of the tumor by 2 months after injection. Other cell lines hadsimilar gene expression phenotypes, but grew more slowly, or requiredhigher numbers of inoculated cells to form tumors. Cells were H-2^(B)haplotype, and expressed high levels of tax.

C. Effect of Oligonucleotides on Tumor Cells In Vitro

Cells of the B line were cultured in T-75 flasks to 70% confluence.Cells were grown in modified DMEM (Cellgro, Mediatech, Washington, D.C.)plus 10% heat inactivated fetal calf serum (FCS) (GIBCO, Grand Island,N.Y.) except where indicated. The unmodified or PS modified ODNs wereadded to a final concentration of 20 μg/cc for 48 hrs. The cells werepre-incubated for 6 hrs in the presence of these ODNs with 10% FCS orwithout serum prior to transfections. Poly A+ RNA was extracted fromtissues and digested with proteinase K and purified by the FastTrackmethod (Bradley, et al., Bio-Techniques, 6:114, 1988) (Invitrogen, SanDiego, Calif.). Samples were denatured and run on formaldehyde 1.4%agarose gels followed by blotting onto nylon membrane and UV fixation.Cloned cDNAs (early genes) were used for generation of probes. Probeswere labeled via the random primer method with ³²P dATP plus dCTP.Specific activities were 1×10⁹ cpm/μg. The effect of antisense on tax orNF-κB/gene expression in vitro is shown by Northern analysis in FIGS. 1,A and B, respectively.

Tax specific sense ODNs used as a control, had no effect on cell growthor gene expression (FIG. 1A lane 2). Previous analyses of tax antisenseODN treated cells demonstrated approximately 10 fold decrease in taxprotein production, which led to a corresponding 10 fold decrease intransactivation of the HTLV-I LTR in transient assays. A specificdecrease in the levels of specific RNA can be seen, and this isconsistent with direct (CRE/ATF) activation of this promoter by tax(Fujii, et al., Proc. Natl. Acad. Sci., 85:8526, 1988). However, noalteration in the levels of many other growth related genes was seen(FIG. 1A lane 3), suggesting that they were not directly activated bytax.

In contrast, inhibition of either the p50 or p65 subunits of NF-κBcaused profound effects on endogenous cytokine gene expression (FIG.1B). IL-6 was affected more than GM-CSF and there was no apparent effecton c-fos, tax, or actin expression. Inhibition of p65 was slightly moreeffective than p50. This result demonstrated specificity of the ODNs forthe NF-κB responsive genes. To quantitate functional effects of thisinhibition, transient CAT expression assays were performed on an NF-κBdependent promoter. The plasmid HIV-CAT was used, as this retroviral LTRcontains 2 copies of an NF-κB target sequence and has shown to be highlyNF-κB responsive (Nabel, et al., Nature, 326:711, 1987).

FIG. 2 shows greater than 20 fold inhibition of CAT expression in thepresence of NF-κB p65 antisense ODNs. In these studies, cells (cell lineB) were pretreated with p65 NK-κB oligonucleotides for 8 hours. Thecells were then transfected with 5 μg of an HIV LTR-CAT construct. CATactivity was analyzed after an additional 48 hours. As an internalcontrol, 5cfig of CMV-β gal (Clontech, San Francisco, Calif.) wasco-transfected and analyzed by MUG assay (MacGregor, et al., Methods inMolecular Biol., 1989). Transfections used Lipofectin (Bethesda ResearchLaboratories, Bethesda, Md.) as per Flegner, et al., Proc. Natl. Acad.Sci. 85:7413, 1989). The level of CAT activity was determined by thinlayer chromatography using ¹⁴C chloramphenicol. Results were quantitatedby scintillation counting of the extracted radiographic spots and areshown.

The findings above were further confirmed by direct assay of proteinbinding to NF-κB target sites. Electrophoretic mobility shift analysis(EMSA) of nuclear extracts obtained from unmanipulated, sense andantisense treated cells are shown in FIG. 3.

The target sequence for EMSA consisted of a double stranded 35-mer:5′AGCTTCAACAGAGGGGACTTTCCGAGAGGCTCGAG3′ (ODN A). The underlined sequenceis identical to the NF-κB consensus in mouse Ig k light chain and tothat used by N. Kabrun, et al., Proc. Natl. Acad. Sci., 88:1783, 1991.The second strand was synthesized by hybridization with an 11-mer (ODNB), (complementary to the sequence not underlined) which was labeled atits 3′ end by klenow mediated extension in the presence of α-³²P dATP.This yielded a double stranded labeled 35-mer. The sequence for themutant NF-κB was generated from a 35-mer (ODN c) sequence:5′AGCTTCAACGAGG-CGACTTTCCGAGAGGCTCGAG3′. The NF-κB site is underlinedwith the mutation in small letters. This ODN was hybridized with ODN Band filled using klenow in the presence of unlabeled dNTPs.

For these experiments, 3 μg of nuclear extract from untreated (no),sense treated (se), or antisense treated (an) cells (cell line B) wereincubated with a double stranded ODN encoding the NF-κB consensus targetsequence (Kabrun, et al., Proc. Natl. Acad. Sci., 88: 1783, 1991). 1×10⁴cpm (approx. 0.86 ng) of labeled ODN was used. Procedures were aspublished (Kabrun, et al., Proc. Natl. Acad. Sci., 88: 1783, 1991).Three NF-κB specific bands (I, II, III) (Baldwin, et al. Mol. Cell.Biol., 11:4943, 1991) were readily identified. Competition assays areshown in the left panel. The ratios of molar excess of unlabeledconsensus ODN (NF-κB) or mutant NF-κB(M) are shown.

Analysis of unmanipulated cells (lanes 1,6) revealed 3 bands of NF-κBcomplexes similar to those previously described (A. Baldwin, et al.,Mol. Cell. Biol., 11:4943, 1991). Unlabeled NF-κB consensusoligonucleotide competitively inhibited all complexes (lanes 2,3). Incontrast, an oligonucleotide with a single G to C substitution in thebinding site failed to block the formation of complexes (lanes 4,5).These data confirm the specificity of this EMSA assay for detection ofNF-κB complexes. Treatment of these cells with p65 sense ODNs had noeffect on complex formation (lane 7). In contrast, use of p65 antisenseODNs specifically ablated all complex formation (lane 8). This occurredin a dose dependent manner and was also obtained with p50 specific ODNs(data not shown).

The effects of tax or NF-κB antisense ODN inhibition on in vitro growthof mouse cell lines are shown in FIG. 4A. Balb/3T3 cells, or cell line Bwere cultured in 10 cm dishes, in the presence of varying concentrationsof FCS (as indicated on the abscissa). 1×10⁴ cells/ml were plated, anamount calculated to give approximately 30% confluence. 20 μg/ml p65sense or antisense ODNs were added and cultures were allowed to grow for6 days. ODNs were replenished at 3 day intervals. Cells were counted andexpressed as % confluence. Mock treatment (No; >), sense (SEN; O) andantisense (ANT; •) are indicated. For each cell line, mock, sense andantisense PS ODN treatments were performed. A constant amount of ODN (20μg/ml) was used, and growth rates were measured daily. The data in FIG.4 shows the percent confluence at day 6 as a function of varying serumconcentration. Serum concentration has profound effects on the growthrate of cells and previous studies have demonstrated induction ofnuclear translocation of NF-κB by serum (T. A. Libermann, et al., Mol.Cell. Biol., 10:2327, 1990). Therefore, this allowed the effects of ODNsto be displayed over a wide range of growth conditions. Treatment withtax specific ODNs, which caused a 90% inhibition of tax expression, hadno apparent effects on growth rates of cells. In contrast, p65 antisenseODNs had profound effects on the growth of cell line B, with no apparenteffect on Balb/3T3 growth at all serum concentrations. Similar resultswere obtained when the concentration of NF-κB ODN was varied and serumconcentration was fixed, or with other tax expressing fibrosarcomalines. p50 ODNs were slightly less effective at the same concentrationbut gave a similar profile. Mixtures of both p50 and p65 ODNs had anadditive inhibitory effect.

It is likely that additional HTLV-I encoded proteins other than tax arenecessary for transformation by the native virus. Thus, tax and NF-κBmay play different roles in maintenance of transformation of humanlymphocytes than that demonstrated for mouse fibroblasts. To determinethe effects of tax and NF-κB antisense ODNs on lymphocytes, the HTLV-1transformed human cell line, MT2 (M. Kozac, Nature, 308:241, 1984) wasanalyzed. FIG. 2B shows a profile of growth inhibition similar to thatseen for the HTLV-1 transformed murine cell line MT-2(Kozac, Nature,308:241, 1983). Cells were grown in 4% FCS in the presence or absence ofNF-κB or tax ODNs. Synthesis of new PS ODN was required, as the 5′ endof the tax translation initiation target sequence in the transgenic micevaried slightly from that in the native human virus. (The sequence forinhibition of tax in human cells is antisense5′-TCGTCTGCCATG-GTGAAGAT-3′.) Cells were allowed to grow for up to 20days. Growth is expressed as absolute cell number in triplicate 6 welldishes. Tax antisense ODNs had no apparent effect on growth of theinfected human cell line (left panel), despite significant inhibition oftax protein. In contrast, p65 antisense ODNs profoundly inhibited growthat all time points.

Example 2 Antisense Inhibition of Tumor Growth In Vivo

Studies were performed on the effects of ODN mediated suppression ofunmanipulated line B cells which were transferred to syngeneic C57B1/6mice. Once tumors were established (7 days after injection in the hindlimb), mice were treated with 3 intraperitoneal (IP) injections at 3 dayintervals of 40 μg/g of sense and antisense ODNs as described byGoodchild, et al. (T. A. Libermann, et al., Mol. Cell. Biol., 10:2327,1990). Western blot analysis of tumors exposed to tax antisense ODNtreatment showed virtually complete suppression of tax expression at the40 μg/g dose. As previously seen above in in vitro studies, noperturbation in growth rate of tumors was seen in the animals treatedwith tax antisense ODNs. Treatment with antisense ODN to NF-κB haddramatically different effects. FIG. 5 shows a typical comparison oftypical matched mice either 15 days after treatment with sense (left) orantisense (right) ODNs to p65. In each case, treatment with antisense top65 caused regression of tumors, while treatment with sense caused nodiminution in growth. A total of eight antisense treated and eightcontrols were analyzed over time, and growth rates were determined byweighing excised tumors. The results are shown in Table 1.

TABLE 1 TIME COURSE ANALYSIS OF NF-_(K)B p65 SENSE AND ANTISENSE TREATEDMICE DAYS AFTER START TREATMENT OF TREATMENT NONE SENSE ANTISENSE 0  348± 214^(a) N.D.^(b) N.D. 4  799 ± 343  748 ± 424 260 ± 198 8 1385 ± 7741264 ± 671 122 + 84 15 5394 ± 2864 4874 ± 2571  45 ± 28 60 N.D. 7243 ±3872 <45^(c) ^(a)tumor weight without capsules (mg) ^(b)not determined⁰undetectable (less than 45 mg)

Clear growth inhibition was seen as early as 8 days after firsttreatment with profound differences occurring by 15 days. Histologicanalysis of these tumors was also performed. Samples obtained from micetreated with sense NF-κB ODN revealed typical morphology characteristicof growing tumors. Treatment with antisense ODN showed focal tumornecrosis with inflammatory infiltrate by 4 days, followed by widespreadsegmental necrosis by 7 days. By 10 days, fibrotic tissue and tumorcapsule with occasional inflammatory cells were all that remained.Beyond 10 days, it was difficult to identify the tumor site. Untreatedmice, or those treated with sense ODNs, died between 8 and 12 weeks,while antisense ODN treated mice have been followed for up to 5 monthswithout evidence of recurrence of tumors. In none of these mice were ODNinjections given beyond the first 9 days.

The present studies reveal striking similarities between the effects oftax on mouse fibroblasts and HTLV-I or II virus on human T-cells. Thoughtax is necessary for transformation of mouse fibroblasts or humanT-cells, the present studies indicate that continued high levels of taxexpression (greater than 10% of the unmanipulated tumor) is notnecessary for maintenance of the activated phenotype, or for growth ofthese cells. Similarly, HTLV-I associated human lymphomas frequentlyexpress very low levels of tax (T. Kinoshita, et al., Proc. Natl. Acad.Sci. USA., 86:5620, 1989). In contrast, tumor growth is very sensitiveto the levels of NF-κB expression. It appears that fully transformedmouse or human cells take up sufficient ODNs to effect tumor regressionat doses of ODN which are well tolerated in vivo. In fact, previoustoxicity studies suggest that mice readily tolerate at least 2.5 timesamounts used here (J. Goodchild, et al., Proc. Natl. Acad. Sci. USA.,85:5507, 1988). The basis for this difference in susceptibility of taxtransformed and normal cells to NF-κB ODNs is unclear. It may representa true difference in the requirement of intracellular NF-κB forsurvival. Alternatively, it may reflect a difference in the ability tointernalize the ODNs. Previous studies have correlated theactivation/growth state of cells with their ability to take upoligonucleotides (R. M. Crooke, Anti-Cancer Drug Design, 6:609, 1991; P.Iversen, ibid 6:531, 1991), and this process appears to occur viareceptor mediated endocytosis. Tax or virus transformed tumor cellsappear highly activated, which may cause them to take up more ODNs.Preliminary data supports this possibility.

Histologic analysis reported here showed profound and widespread tumornecrosis, and even this short window of treatment was apparentlysufficient to prevent tumor recurrences. This suggests that these ODNsmay provide a valuable approach to therapy of HTLV-I associated adultT-cell leukemia which has proved largely refractory to other modalities.In addition, these antisense sequences should be able to inhibit otherprocesses which rely upon activation of NF-κB.

Example 3 Antisense NF-κB ODNs Prevent LPS Mediated Death

In order to compare the effects of NAC or NF-κB AS-ODNs on the toxicmanifestations of LPS induced septic shock, survival rates for thedifferent treatment groups of mice were compared. This data issummarized in FIG. 6.

LPS-induced lethality. In vivo LPS challenge was performed according tothe modified methods of Broner et al, Critical Care Medicine 16:848,1988; and Peristeris, et al., Cellular Immunology, 140:390, 1992. Atotal of 35 C57BI/6 mice (body weight 25-35 g) were studied in threetreatment groups: 1)10 mice were treated with LPS followed by two mockinjections of saline. 2) 10 mice were pretreated with NAC 12 hours and ½hour prior to inoculation with LPS. 3) 10 mice were pretreated withNF-κB antisense ODNs 20 hours and ½ hour prior to LPS, and 5 mice weretreated 20 and ½ hours prior to LPS with an irrelevant HTLV-I specificantisense ODN (Kitajima, I., et al., J. Biol. Chem. 267: 25881, 1992) asa control.

LPS (Escherichia coli 055:B5, Sigma, St. Louis., MO) was given as asingle dose by intraperitoneal (ip) injection. Optimal results wereobtained when a total dose of 100-150 mg (approx 5 mg/g body wt.) wasadministered in 0.3 ml of sterile, pyrogen-free saline. Qualitativelysimilar results were obtained when sepsis was induced with 75 or 200 mg,with delayed or accelerated sickness, respectively. Two experiments wereperformed with 5 mice per group. NAC (Sigma, St. Louis Mo.) was givenintraperitonealy at a dose of 0.27 mg/g of body weight at a single site.Antisense NF-κB p65 ODN (sequence 5′-AAACA-GATCGTCCATGGTCA-3′) was 3′terminal phosphorothioate (PS)-modified (Kitajima, I., et al., Science,259:1523, 1993; and Winer, B. J. 1971. Statistical Principles InExperimental Design. McGraw-Hill New York, N.Y.), as this has been shownto confer stability in animals (Kitajima, I., et al., J. Biol. Chem.267:25881, 1992). Extensive previous experience with this ODN, incomparison with NF-κB sense and irrelevant antisense controls has shownit to be highly specific, both in vivo and in vitro (Kitajima, I., etal., Science 258:1792, 1992). The p65 AS ODN preserves this specificityeven when used at doses twice the concentration used in this study.Antisense ODNs were administered ip at a dose of 40 mg/g of body weight,as it has been demonstrated that in previous in vivo studies (Kitajima,I., et al., J. Biol. Chem. 267:25881, 1992) this is an effective dose.AS-ODNs were administered i.p. in a volume of 0.3 ml sterile saline,while no treatment controls received only 0.3 ml of sterile saline. Thenumber of surviving mice in each treatment group was recorded beginningat 12 hrs and additional observations were performed at 24, 48, 72 and96 hours. The significance of differences in the survival rates betweengroups was evaluated by a Chi square test using all data points otherthan zero time. This provides an extremely conservative analysis inwhich a p value less than 0.05 is highly significant (Winer, B. J. 1971.Statistical Principles In Experimental Design. McGraw-Hill New York,N.Y.).

Control mice injected with LPS only, became ill within a few hours. Thiswas manifested as decreased spontaneous activity, followed by hunchingand decreased oral intake. Two of ten mice died by 12 hours and all ofthese mice ultimately died by 48 hours after a single intraperitonealinjection of 5 mg/g body wt (100-150 mg) (solid bars). Mice which werepretreated with NAC prior to LPS challenge showed increased but notsignificant protection from lethality (Total Chi Square 8.31, p=0.08),in agreement with previous studies (Peristeris, P., et al., CellularImmunology 140:390, 1992; Broner, C. W., et al., Critical Care Medicine16:848, 1988) (striped bars). Ultimate survival rate was 40% with onedeath occurring prior to 12 hours. Mice pre-treated with antisense NF-κBODNs had a significantly better outcome (Total Chi square 11.86 p=0.018)(dotted bars). Deaths were delayed beyond 12 hours and the ultimatesurvival rate was 70%. Treatment with tax antisense or NF-κB sensecontrol ODNs (Kitajima, I., et al., Science. 258:1792, 1992; Kitajima,I., et al., J. Biol. Chem. 267:25881, 1992) gave results identical tothe untreated control.

Example 4 Serum Levels of IL-6 are Decreased by NF-κb Antisense ODNswhen Administered Prior to Septic Shock

The results of Example 3 suggest NF-κB antisense inhibition dramaticallyreduces LPS induced lethality. In order to confirm that this is aspecific consequence of NF-κB inhibition, the level of serum IL-6 wasmeasured. Previous studies have demonstrated that IL-6 serves as anaccurate indicator for NF-κB activation as this is a primarytranscriptional activator of this cytokine (Liberman, T., et al., Cell.Biol. 10:2327, 1990). In addition, IL-6 is an important mediator ofcatastrophic immune responses such as inflammatory nephropathy (Horii,Y., et al., J. Immunol. 143:3949, 1989; Rugo, H. S., et al., J. Clin.Invest. 89:1032, 1992), may modulate immunoreactivity of tumors(Tabibzadeh, S. S., et al., Am. J. Pathol. 135:1025, 1989), and has beenstrongly implicated in the pathogenesis of septic shock (Ulich, T. R.,et al., J. Immunol 146:2316, 1991; Troutt, A. B. et al., J. CellularPhysiology, 138:38, 1989).

Assay for IL-6 protein in serum. Sera from mice within each treatmentgroup were obtained at 0, 1, 4, 8, and 20 hrs after LPS administration.Serum IL-6 was determined by an enzyme-linked immunosorbent assay (EUSA)according to the method of Pruslin et al. (Pruslin, F. H., et al., J.Immunol. Method 137:27, 1991) using rat anti-mouse monoclonal antibodiesto IL-6 (Pharmingen, San Diego, Calif.). The detection limit for thisassay was 100 pg/ml. Statistical differences between treatment groupswere analyzed by the ANOVA test for time points beyond zero. Statisticalanalysis was performed using the program Statview, on a Macintoshcomputer.

Serum IL-6 protein levels were measured by ELISA. Three mice were usedper treatment group and experiments were repeated twice.Inter-experiment variability was less than 20%. Data were pooled fromthese experiments and are displayed in FIG. 7 (No Rx, LPS given at timezero without specific inhibitor. NAC, pretreatment as described inmethods. NF-κB, pretreatment with ODN. No LPS, mice given sterile salineonly at corresponding times). IL-6 levels rapidly rose from the lowerlimit of detectability (100 pg/ml) at time zero to peak levels (5800pg/ml) at 4 hrs after ip injection of LPS. Levels remained high (3900pg/ml) up to 20 hrs after treatment. In contrast, serum levels of IL-6were almost two fold (51%) lower at 4 hrs after LPS challenge (2900pg/ml) in mice pretreated with antisense NF-κB ODNs. The serum IL-6levels at 4 hours in mice pretreated with NAC were not significantlydifferent from the LPS only control (74% or 4300 pg/ml) (FIG. 7).

ANOVA analysis of the 3 groups at 4, 8 and 20 hours, revealed areduction of IL-6 levels by NAC which was barely significant [p=0.03,F(1,24)=6.68], and a highly significant reduction by NF-κB antisense ODN[p=0.0001, F(1,24)=71.-61].

Example 5 Tissue Specific Effect of NF-κB Antisense ODNs on LPS InducedIL-6 mRNA

Previous studies in mice have suggested nonuniform accumulation ofoligonucleotides in different tissues when administeredintraperitoneally or intravenously (Iversen, P. et al., Anti-cancer DrugDesign 6:531, 1991). Previous studies suggested that oligonucleotideuptake by cells may be highly dependent on activation state (Kitajima,I., et al., J. Biol. Chem. 267:25881, 1992). Therefore, the effects ofNF-κB antisense on IL-6 in individual tissues in the presence or absenseof LPS activation were evaluated. IL-6 mRNA expression was monitoredbecause previous studies have shown that its expression may bepleomorphically induced in a large number of tissues (Ulich, T. R., etal., J. Immunol 146:2316, 1991; Troutt, A. B. et al., J. CellularPhysiology, 138:38, 1989) and can therefore be used to assess NF-κBinhibition in variety of tissues.

Northern blot analysis. Kidney, liver, lung, spleen and salivary glandsfrom representative mice of each group were rapidly frozen in liquidnitrogen and crushed with a sterile pestle while frozen, mRNA wasextracted using the Fast Track method (Bradley, J. E., et al.,BioTechniques 6:114, 1988) (Invitrogen, San Diego, Calif.). The positivecontrol for murine gene expression (TNF-a, IL-6, MHC class I andGM-CSF), was mRNA obtained from an HTLV-I tax expressing mousefibrosarcoma cell line (Kitajima, I., et al., J. Biol. Chem. 267:25881,1992). 7 mg mRNA per lane was loaded and electrophoresis was performedon formaldehyde 1.2% agarose gels. Samples were blotted onto nylonmembrane and fixed by UV crosslinking. Murine cDNA probes of MHC class I& II and GM-CSF were labeled via the random primer method with[³²P]dATP. IL-6 TNF-α and actin probes cloned in pUC, pBluescript orpgem plasmids were labeled with [³²P]dATP by polymerase chain reactionusing universal primers complementary to regions flanking the linkers.Specific activities of probes were in excess of 1X10⁹ cpm/mg.

mRNAs were extracted from each tissue, 3 hrs after LPS injection. InFIG. 8, the lanes were as follows: B is an HTLV-I tax transformed cellline which expresses high levels of NF-κB and serves as a positivecontrol. No is obtained from mock (saline treated) mice. LPS, obtainedafter no pretreatment. NAC, is from pretreated mice (8 mg×2). NF is froman animal pretreated with 40 mg/g NF-κB antisense ODN. DNA probes (IL-6,TNF-α, MHC class I, GM-CSF and actin) are indicated at the left.Analysis of liver and kidney are shown in panel A and lung, salivarygland and spleen are shown in panel B. Lanes 1-9 represent pretreatmentfor 3 hours with NAC or antisense NF-κB ODN. Lanes 10-18 show theeffects of 24 hrs pretreatment.

Pretreatment by antisense NF-κB ODNs caused a profound decrease in LPSinduced IL-6 mRNA in kidney (FIG. 8A, compare lanes 18 to 16 and 9 to7), with somewhat less effect in lung (FIG. 8B compare lanes 5 to 3),and lower effect in liver or other tissues. The uptake and inhibitoryeffect of the oligonucleotides was time dependent as effects were muchgreater when ODNs were administered 20 hrs rather than 3 hrs prior toLPS (compare right and left hand portions of FIG. 8A). NAC pre-treatmentinhibited IL-6 expression in a qualitatively similar manner but was lesseffective in most tissues tested.

To further confirm the specificity of NF-κB inhibition in tissues, mRNAof two additional genes were analyzed, TNF-α and MHC class I, both knownto be NF-κB dependent and implicated in the pathogenesis of sepsis orcatastrophic immune responses. Both antisense and NAC down-modulatedexpression of these genes though the effect was less than that for IL-6(FIG. 8). FIGS. 8 A and B show that IL-6 mRNA expression was highlyinduced by LPS in liver, kidney and spleen, moderately induced in lungand weakly induced in salivary gland; IL-6 expression was not detectedin brain and muscle.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade without department from the spirit or scope of the invention.

1. A method of treating cancer in a subject comprising administering toa subject a therapeutically effective amount of an oligonucleotide thatinhibits NF-κB expression, thereby treating the cancer.
 2. A method oftreating cancer in a subject comprising administering to a subject atherapeutically effective amount of an oligonucleotide that contains aCpG motif, thereby treating the cancer.
 3. The method of claim 1 or 2,wherein the cancer is a solid tumor.
 4. The method of claim 1 or 2,wherein the cancer is a cancer of B-cells, T-cells or fibroblast cells.5. The method of claim 4, wherein the T-cell cancer is leukemia.
 6. Themethod of claim 1, wherein the oligonucleotide contains a phosphatebackbone that is chemically modified by substitution of a non-bridgingoxygen atom of the nucleic acid backbone, wherein said substitutiongenerates a backbone modification comprising a moiety selected from thegroup consisting of methane phosphate and methyl phosphate.
 7. Themethod of claim 6, wherein the substitution is at one or morenucleotides at the 3′ terminus, the 5′ terminus, or both terminii of theoligonucleotide.
 8. The method of claim 1, wherein the oligonucleotideis DNA.
 9. The method of claim 1, wherein the oligonucleotide is RNA.10. The method of claim 1, wherein the oligonucleotide has a sequencecomprising at least an 8 nucleotide fragment of any one of SEQ ID NO:5,6, 7, or 8, wherein the oligonucleotide contains not more than 40nucleotides.
 11. The method of claim 1, wherein the oligonucleotide isselected from the group consisting of SEQ ID NO:5, 6, 7, and
 8. 12. Themethod of claim 1, wherein the oligonucleotide is administered in anaqueous or non-aqueous vehicle.
 13. The method of claim 1, wherein theoligonucleotide is administered in combination with a glutathioneprecursor.